The molecular characterisation of the vernalisation response in safflower via the development of genomic and transcriptomic resources

Safflower (Carthamus tinctorius) is an oilseed grown globally. There is an interest in modifying safflower to cope with climate change and to adapt to new agronomic trends. While almost all cropped safflower are spring varieties, a number of wild safflower varieties are noted as ’winter hardy’. These display characteristics found in plants that respond to vernalisation (an extended period of non-freezing cold). Because flowering traits, including the vernalisation response, are linked to yield and adaptability to climate, this PhD project sought to understand the vernalisation response in safflower, as a component of flowering time. The vernalisation response in ’winter hardy’ and spring safflower was investigated. It was confirmed that winter safflower does respond to vernalisation conditions, similar to other plant species. The vernalisation response in winter safflower is saturated after approximately 2 weeks exposure to vernalisation conditions. It is inheritable, epigenetic and appears to be dependent on a single recessive allele, as shown by segregation ratios in a crossing population created from winter and spring parents. Two approaches were developed to characterise this vernalisation response. Firstly, RNA sequencing (RNA-Seq) was performed on RNA extracted from winter and spring safflower before, during and after exposure to vernalisation conditions. Differential expression analyses on the resulting RNA-Seq datasets tentatively identified four genes as directing functional roles in the vernalisation response of safflower: APETALA 1-LIKE (CtAP1-LIKE), MADS-BOX DOMAIN CONTAINING 1 (CtMADS1), FLOWERING LOCUS T-LIKE (CtFT-LIKE) and VERNALISATION 1-LIKE (CtVRN1-LIKE). This analysis also identified 33 additional gene products (annotated transcripts or transcripts of unknown function) as candidates for further experimental investigation. In addition to the transcriptomic data, genomic resources were developed to further characterise the molecular basis of the vernalisation response. A high quality de novo assembly was constructed using Illumina reads from spring safflower, covering approximately 80% of the estimated 1,400,000,000 base pair (1.4 Gbp) spring safflower genome. Using this draft genome in combination with F3 crossing families and a genetic marker approach, 27 genetic markers for vernalisation were identified. Furthermore, these markers were mapped to a recent genetic map of safflower (Bowers 2016), clustering in close proximity to one another on chromosome 8. A single differentially expressed transcript, identified in the transcriptomic analyses, was located on the same chromosome. However, the transcript of interest was mapped to a chromosome 8 position some distance away from the identified markers. These high quality transcriptomic and genomic resources were used to identify the molecular basis for vernalisation in safflower. The investigative approaches developed in this project can also be utilised to characterise the molecular mechanisms of other traits in safflower.